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SUPPORTING INFORMATION

Plasma Concentration Gradient Influences the Protein Corona Decoration on Nanoparticles

Mahdi Ghavamia, Samaneh Saffarb, Baharak AbdeEmamya, Afshin Peirovib, Mohammad A.

Shokrgozara, Vahid Serpooshanc, and Morteza Mahmoudic,d,e*

aNational Cell Bank, Pasteur Institute of Iran, Tehran, Iran.

bCore Facility Center, Pasteur Institute of Iran, Tehran, Iran.

cDivision of Pediatric Cardiology, Department of Pediatrics, Stanford University School of

Medicine, Stanford, California 94305-5101, United States

dNanotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical

Sciences, Tehran, Iran.

eCurrent Address: School of Chemical Sciences, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801

*Corresponding Author: Web: www.biospion.com; Email: [email protected]

Electronic Supplementary Material (ESI) for RSC AdvancesThis journal is © The Royal Society of Chemistry 2012

Experimental Section

Materials

Nanoparticles (NPs). Carboxylated polystyrene NPs with the mean size of 100 nm were

purchased from Invitrogen (USA). Plain silica NPs with 2 different sizes (100 and 200 nm)

were purchased from Kisker-Biotech inc. (Germany).

Human plasma.Blood was withdrawn from 10 volunteers from the National Cell Bank staff

(Pasteur Institute of Iran, Tehran, Iran), and collected into 10 ml K2EDTA coated tubes (BD

Bioscience (UK)). Plasma was prepared following the HUPO BBB SOP guidelines.[1]

Hard corona preparation. Samples were incubated under non-gradient plasma (i.e. 10%,

30%, 50%, 70%, and 80%) and gradient-plasma (i.e. 10%-30%, 50%-70%, and 70%-80%)

concentrations. Plasma solutions were diluted with PBS maintaining the final NP

concentration constant and equal to 100 μg/ml. For the non-gradient plasma interactions, the

NPs were incubated with the plasma solutions at 37°C for one hour. For the gradient plasma

interactions, the NPs were incubated with the initial plasma solutions at 37°C for half an hour

followed by an additional half an hour incubation with increased plasma (pure plasma was

added to the incubated solution to increase the protein concentration). To obtain hard corona

complexes, after the incubation in various types of plasmas, NPs were centrifuged to pellet

the particle-protein complexes and separated from the supernatant plasma, as recognized as

standard method[2]. The pellet was then re-suspended in 500 μl of PBS and centrifuged again

for 3 minutes at 18 krcf at 4°C to pellet the particle-protein complexes. The standard

procedure consisted of three washing steps before re-suspension of the final pellet to the

desired concentration.

SDS PAGE. Upon completion of the last centrifugation step, the NP-protein corona pellet

was re-suspended in protein loading buffer and boiled for 5 minutes at 100 °C. Subsequently,

an equal sample volume was loaded in 15% and 20% polyacrylamide gels and gel


electrophoresis was performed at 120V, 400mA for about 60 minutes (for each sample), until

the proteins neared the end of the gel. The gels were stained with silver staining method.

Liquid chromatography mass spectrometry (LC-MS/MS) technique.Associated proteins

in the hard corona composition of the desired bands (Figure 3 b and d) were probed.The

bands were cut and the proteins were collected. Samples were loaded onto a nanoAcquity

UPLC system (Waters Corp., USA) equipped with a nanoAcquity Symmetry C18, 5 µm trap

(180 µm x 20 mm Waters) and a nanoAcquity BEH130 1.7 µm C18 capillary column (75 m

x 250 mm, Waters). The trap wash solvent was 0.1% (v/v) aqueous formic acid and the

trapping flow rate was 10 µL/min. The trap was washed for 5 min before switching flow to

the capillary column. The separation used a gradient elution of two solvents (solvent A:

0.1% (v/v) formic acid; solvent B: acetonitrile containing 0.1% (v/v) formic acid). The flow

rate for the capillary column was 300 nL/min. Column temperature was set at 60°C and the

gradient profile was as follows: initial conditions of 5% solvent B, followed by a linear

gradient to 30% solvent B over 125 min, then a linear gradient to 50% solvent B over 5 min,

followed by a wash with 95% solvent B for 10 min. The column was returned to initial

conditions and re-equilibrated for 30 minutes before subsequent injections. The nanoLC

system was interfaced with a maXis LC-MS/MS System (BrukerDaltonics) with a nano-

electrospray source fitted with a steel emitter needle (180 µm O.D. x 30 µm I.D., Proxeon).

Positive ESI- MS & MS/MS spectra were acquired using AutoMSMS mode. Instrument

control, data acquisition and processing were performed using Compass 1.3 SR3 software

(microTOF control, Hystar and DataAnalysis, BrukerDaltonics). Instrument settings were:

ion spray voltage: 1,500 V, dry gas: 6 L/min, dry gas temperature 160 °C, ion acquisition

range: m/z 50-2,200. AutoMSMS settings were: MS: 0.5 s (acquisition of survey spectrum),

MS/MS (CID with N2 as collision gas): ion acquisition range: m/z 300-1,500, 0.1 s


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where NpSpCk is the normalized percentage of spectral count for protein k, SpC is the

spectral count identified, and Mw is the molecular weight (in kDa) of the protein k. Using

equation 1, the protein size can be calculated and the actual contribution of each protein to

the hard corona composition can be evaluated.[4] In order to determine the change in the

protein amount, the equation below was used:

/ (2)

The values below 0.6 (60%) were considered as significant decrease in protein amount and

presented in Table 1.


Differential Centrifugal Sedimentation (DCS).DCS experiments were performed usinga

CPS Disc Centrifuge DC24000. The Disc Centrifuge device is a particle size analyzer for

measuring particles in the range of 0.01µm to 40µm. The analyzer measures particle size

distributions using centrifugal sedimentation within an optically clear spinning disc that is

filled with fluid. Sedimentation is stabilized by a density gradient within the fluid and the

accuracy of the measured size is insured through the use of a known size calibration standard

run before each measurement. The use of a biological sample with a large amount of proteins

requires a new sucrose gradient to be prepared for each measurement. Concentration of the

particles at each size is determined by continuously measuring the turbidity of the fluid near

the outside edge of the rotating disc. The turbidity measurements are converted to a weight

distribution by Mie theory light scattering calculations. The choice of the experimental

parameters such asfluid gradient and the speed of rotation is based on the type of arterial

being analyzed and the range of sizes being measured. The primary information from the

analytical disk centrifuge is the time taken for the particles to travel from the centre of the

disk through a defined viscous sucrose gradient to a detector placed at the outer rim of the

disk under a strong centrifugal force. For materials with homogenous density and simple

shape (.g. spherical particles) this time can be directly related to theparticle size. Whenobjects

are inhomogeneous, or irregular in shape, the different arrival times still allow to distinguish

between the particles. In the simplest approximation, it is possible to approximate them to an

equivalent uniform sphere. This was applied to the entire figures presented in this work, by

citingthe sizes on the x-axis; hence, in the case of aggregated particles (e.g. dimers and

trimers), the cited size should be considered as the‘apparent’ size for particles. Moreover, for

the sake of clarity in the comparisonsmade between different samples, we chose to present

the data as relative weight particle size distribution. The tallest peak (highest weight value) in

the distribution is called the ‘base’ peak (has a value of 100%) and all other particle size


(multimer) peaks are then normalized against this base peak to give a relative weight

distribution. It is noteworthy that the conversion from absorption raw data to molecular

weight data is correct as long as the optical parameters and the density for particles and fluid

are correct and the particles are spherical. In this regard, the information we obtainforthe

large NP-protein clusters at about 500 nm are merely indicative of their existence but do not

provide insight totheir actualsize or their absolute amount. It is notable that different sucrose

gradient (1.018g/ml and 1.064g/ml) were employed for polystyrene and silica particles,

respectively.


Gel results for 200 nm silica particles

(a) (b)

(c) (d)

Figure S1: (a) 15% and (b) 20% SDS-PAGE gel of human plasma proteins obtained from silica(200 nm in size)-protein complexes free from excess plasma following incubation with human plasma at various concentrations (both non-gradient and gradient situations); from left to right, the lanes stand for protein ladder, 10%, 30%, 10%-30%, 50%, and 30%-50% . (c) 15% and (d) 20% SDS-PAGE gel similar to (a) and (b) with different plasma concentration from left to right, the lanes stand for protein ladder, 50%, 70%, 50%-70%, 80%, and 70%-80%


DCS results for 200 nm silica particles

Figure S2: DCS results for silica (200 nm) nanoparticles interacted with human plasma

proteins at various non-gradient and gradient circumstances in situ; right graph is the

enlargement of the main peak areas of the DCS graphs to enhance the shift to smaller size

after interaction with plasma proteins.


References [1] A. J. Rai, C. A. Gelfand, B. C. Haywood, D. J. Warunek, J. Yi, M. D. Schuchard, R. J. Mehigh, S.

L. Cockrill, G. B. I. Scott, H. Tammen, P. Schulz‐Knappe, D. W. Speicher, F. Vitzthum, B. B. Haab, G. Siest, D. W. Chan, PROTEOMICS 2005, 5, 3262‐3277.

[2] M. P. Monopoli, D. Walczyk, A. Campbell, G. Elia, I. Lynch, F. BaldelliBombelli, K. A. Dawson, Journal of the American Chemical Society 2011, 133, 2525‐2534.

[3] M. P. Monopoli, D. Walczyk, A. Campbell, G. Elia, I. Lynch, F. BaldelliBombelli, K. A. Dawson, Journal of the American Chemical Society 2011, 133, 2525‐2534.

[4] D. Walczyk, F. B. Bombelli, M. P. Monopoli, I. Lynch, K. A. Dawson, Journal of the American Chemical Society 2010, 132, 5761‐5768.

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